PROJECT SUMMARY Glycans have several distinct properties that make them excellent targets for disease biomarkers. Firstly, their location glycans on cell surfaces makes them the first point of contact for cellular interactions, and thus they are crucial in the control of normal metabolic processes. Secondly, cell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cells. Thirdly, specific glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states, such as cancer. And lastly, changes in glycosylation involve many proteins, including those that are highly abundant. Therefore, a single change in a cell's glycosylation machinery can affect many different glycoconjugates. To effectively employ and discover glycan disease markers a wide range of highly-specific reagents are urgently needed. Using structurally-guided directed evolution, we will convert the newly identified PNGase F-II carbohydrate- processing enzyme into a pan-specific high-affinity reagent for peptides and proteins that contain asparagine- linked (N-linked) carbohydrate chains. Such engineered lectin-like reagents derived from enzymes are called ?Lectenz?, and have several advantages over lectins and antibodies. The advantages of Lectenz include precise definition of specificity, ease of recombinant expression, and (for human enzyme homologues) minimal in vivo toxicity, enabling their potential use as imaging reagents. A pan-specific N-glycan Lectenz derived from PNGase F-II would directly address the needs of glyco-biomarker detection in mass spectrometry-based glycomics/proteomics analysis by enabling sample enrichment. Glycopeptide sample enrichment aids glycosylation site-mapping by eliminating non-glycosylated peptides, which would otherwise lower the signals from glycopeptides that have low ionization efficiency. Glycosylation site mapping is essential in fully characterizing and exploiting glycans as markers of specific disease states, and yet no current reagents exist that can be used to enrich a sample in all constituent N-linked glycans. The principle advantages of an engineered Lectenz over an antibody are that the Lectenz is specific to the carbohydrate sequence, but, in contrast to antibodies, will recognize that sequence in a broad range of glycans. Further, in contrast to carbohydrate reagents based on plant lectins, engineered Lectenz are derived from enzymes that have exquisite substrate specificities and low toxicities.